1.1 Field of the Invention
The present invention relates to amphiphilic oligomer conjugates capable of traversing the blood-brain barrier (xe2x80x9cBBBxe2x80x9d) and to methods of making and using such conjugates. The conjugates of the invention comprise therapeutic agents such as proteins, peptides, nucleosides, nucleotides, antiviral agents, antineoplastic agents, antibiotics, etc., and prodrugs, precursors, derivatives and intermediates thereof, chemically coupled to amphiphilic oligomers.
1.2 Description of the Related Art
In the field of pharmaceutical and therapeutic invention and the treatment of disease states and enhancement of physiological conditions associated with the CNS, a wide variety of therapeutic agents have been developed, including proteins, peptides, nucleosides; nucleotides, antiviral agents, antineoplastic agents, antibiotics, etc., and prodrugs, precursors, derivatives and intermediates thereof.
Additionally, the many known neuroactive peptides offer additional possibilities for useful therapeutic agents. Such neuroactive peptides play important biochemical roles in the CNS, for example as neurotransmitters and/or neuromodulators. Delivery of this diverse array of peptides to the CNS provides many opportunities for therapeutic benefit. For example, delivery of endogenous and synthetic opioid peptides, such as the enkephalins, can be used to effect analgesia.
However, a number of obstacles currently limit the use of many compounds for use as CNS therapeutic agents.
First, the brain is equipped with a barrier system. The brain barrier system has two major components: the choroid plexus and the blood-brain barrier (BBB). The choroid plexus separates cerebrospinal fluid (CSF) from blood and the BBB separates brain ISF from blood.
The BBB has about 1000 times more surface area than the choroid plexus and is the primary obstacle to delivery of therapeutic compounds to the CNS. The BBB acts as a selective partition, regulating the exchange of substances, including peptides, between the CNS and the peripheral circulation. The primary structure of the BBB is the brain capillary endothelial wall. The tight junctions of brain capillary endothelial cells prevent circulating compounds from reaching the brain ISF by the paracellular route. Furthermore, recent work suggests the existence of a physiological barrier at the level of the basal lamina, in addition to the barrier provided by the tight junctions. Kroll et al., Neurosurgery, Vol. 42, No. 5, p.1083 (May 1998). Other unique characteristics of the BBB include lack of intracellular fenestrations and pinocytic vesicles and a net negative charge on the luminal surface of the endothelium. Id.
The mechanisms by which substances may traverse the BBB may generally be divided into active and passive transport mechanisms. Lipophilic molecules readily traverse the BBB by passive transport or diffusion through the endothelial plasma membranes. In contrast, hydrophilic molecules, such as peptides, typically require Ian active transport system to enable them to cross the BBB. Certain larger peptides, such as insulin, have receptors on the luminal surface of the brain capillaries which act as active transcytosis systems.
Diffusion of many therapeutic compounds, such as peptides, across the BBB is also inhibited by size. For example, cyclosporin, which has a molecular weight of xcx9c1200 Daltons (Da), is transported through the BBB at a much lower rate than its lipid solubility would predict. Such divergence between lipid solubility and BBB permeation rates is probably due to steric hindrances and is common where the molecular weight of a compound exceeds 800-1000 Da.
A further barrier to peptide delivery to the CNS is metabolic instability. In particular, before peptides injected into the blood reach the CNS, they must survive contact with enzyme degrading enzymes in the blood and in the brain capillary endothelium. BBB enzymes are known to degrade most naturally occurring neuropeptides. Orally administered peptides face additional barriers discussed below. Metabolically stabilized peptides may exhibit increased resistance to certain enzymes; however, it has not been possible to protect peptides from the wide range of peptide-degrading enzymes present in the blood and BBB.
Another difficulty inherent in delivering peptides to the BBB is that successful transcytosis is a complex process which requires binding at the lumenal or blood side of the brain capillary endothelium, movement through the endothelial cytoplasm, and exocytosis at the ablumenal or brain side of the BBB. Peptides may bind to the lumenal membrane of the brain capillary endothelium or undergo binding and endocytosis into the intracellular endothelial compartment without being transported into the CNS.
In any event, many currently existing drug substances, especially peptides, are unable to overcome these structural and metabolic barriers to enter the BBB in sufficient quantities to be efficacious. There is therefore a need for pharmaceutical compositions which can (1) withstand degradative enzymes in the blood stream and in the BBB and (2) which can penetrate through the BBB in sufficient amounts and at sufficient rates to be efficacious.
Many attempts have been made in the art to deliver therapeutic compounds, such as peptides, to the CNS with varying levels of success. Such attempts can generally be grouped into two categories: invasive and pharmacological.
Invasive delivery strategies include, for example, mechanical procedures, such as implantation of an intraventricular catheter, followed by pharmaceutical infusion into the ventricular compartment. Aside from general considerations relating to the invasiveness of mechanical procedures, a major difficulty with mechanical approaches is the lack of peptide distribution. For example, injection of peptides into the CSF compartment results in very little distribution beyond the surface of the brain. This lack of distribution is due in part to rapid exportation of peptides to the peripheral circulation.
Another invasive strategy for delivering therapeutic compounds to the CNS is by intracartoid infusion of highly concentrated osmotically active substances, such as mannitol or arabinose. Their high local concentration causes shrinkages of the brain. capillary endothelial cells, resulting in a transient opening of the tight junctions which enable molecules to traverse the BBB. Such procedures have considerable toxic effects, including inflammation, encephalitis, etc. Furthermore, such procedures are not selective: the opening of the tight junctions of the BBB permits many undesirable substances to cross the BBB along with the therapeutically beneficial molecule. For a recent review of osmotic opening and other invasive means for traversing the BBB, see Kroll, Robert A. Neurosurgery, Vol. 42, No. 5, May 1998.
While the risks involved in these invasive procedures maybe justified for life-threatening conditions, they are generally not acceptable for less dramatic illnesses. There is therefore a need for less invasive, non-mechanical and safer means for enabling therapeutic compounds to cross the BBB.
As noted above, lipophilic substances can generally diffuse freely across the BBB. Accordingly, a common pharmacological strategy for enabling peptides to traverse the BBB is to chemically modify the peptide of interest to make it lipid-soluble. Hydrophilic drug substances have been derivatized with short chain or long chain fatty acids to form prodrugs with increased lipophilicity.
Prodrugs are biologically inert molecules which require one or more metabolic steps to convert them into an active form. A difficulty with the prodrug approach to crossing the BBB is that the cleavage necessary to yield an active drug may not occur with sufficient efficiency and accuracy to produce an efficacious amount of the drug.
There is therefore a need for modified stable therapeutic compounds, such as peptides, which are capable of traversing the BBB but which retain all or part of their efficacy without requiring metabolic steps to convert them into an active form.
A further difficulty with lipidized prodrugs is that they pass in and out of the CNS so readily that they may never reach sufficient concentration in the CNS to achieve their intended function. For example, previous attempts have been made to engineer enkephalin conjugates which can traverse the BBB. See Partridge, W. M., xe2x80x9cBlood-Brain Barrier Transport and Peptide Delivery to the Brain,xe2x80x9d Peptide-Based Drug Design: Controlling Transport and Metabolism, p. .277 (1995). However, these strategies required the subcutaneous delivery of frequent and massive doses of peptide to induce analgesia. Frequent and/or massive dosing is inconvenient to the patient and may result in serious side effects.
There is therefore a need in the art for means for enabling therapeutic agents, such as peptides, to cross the BBB in a controlled manner which permits accumulation of sufficient quantities of the therapeutic in the brain to induce the desired therapeutic effect.
Another pharmacological method for delivering peptides across the BBB is to covalently couple the peptide of interest to a peptide for which a specific receptor-mediated transcytosis system exists. xc2x0 For example, it is theoretically possible to attach xcex2-endorphin, which is not normally transported through the BBB, to insulin to be transported across the BBB by insulin receptor-mediated transcytosis. Upon entry into the brain interstitial space, the active peptide (xcex2-endorphin) is then released from the transport vector (insulin) to interact with its own receptor.
However, the difficulty with this system is designing a chimeric molecule which can become detached upon entry into the interstitial space; to the inventor""s knowledge, this has not yet been achieved. Additionally, the poor stoichiometry of the neuropeptide to the carrier molecule limits the mass of the target peptide. Furthermore, receptor-mediated cellular transport systems typically have physiologically limited transport capacity. This is a rate-limiting factor which can prevent entry of pharmaceutically active amounts of-peptide.
There is therefore a need in the art for means for enabling therapeutic substances, such as peptides, to cross the BBB by diffusion so as to avoid the limitations inherent in receptor-mediated transport.
Other pharmacological strategies 7include using an active fragment of a native peptide; modification of a native peptide to increase blood-brain barrier (BBB) transport activity; and delivery of a gene encoding the neuropeptide to the brain.
Oral administration is a desirable and convenient route of administration; however, orally delivered peptides must overcome a series of barriers before they can enter the blood stream. Such peptides must survive proteolysis and the acidic environment of the stomach, gastric and pancreatic enzymes, exo- and endopeptidases in the intestinal brush border membrane.
There is therefore a need for orally administered peptides which can also resist proteolytic enzymes in the blood and BBB and which can traverse the BBB in sufficient quantities to provide broad distribution of drugs into the entire brain parenchyma.
Methionine-enkephalin and leucine-enkephalin are naturally occurring analgesic pentapeptides. These peptides and their analogs are known to-act as neurotransmitters or modulators in pain transmission. Their analgesic properties are short in duration. When administered by intracerebroventricular injection, the duration of their action is also transient.
These properties make the enkephalins attractive compounds for use as therapeutic agents, for mediating analgesia and providing a viable alternative to morphine. However, in order to deliver enkephalkins across the BBB, they must be protected against rapid degration by aminopeptidases and enkephalinases. Furthermore, since enkephalins are hydrophilic peptides, they must be modified to provide them with increased lipophilic characteristics before they can passively diffuse across the BBB into the CNS.
The attractive therapeutic properties of enkephalins have been known for sometime, and many investigators have attempted to enhance the ability of enkephalins to traverse the BBB.
Schroder et al., Proc. Int. Symp. Control Rel. Biact. Material, Vol. 23, p 611 (1996) teaches that Dalargin, a leu-enkephalin analogue can be incorporated in nanoparticles formed by polymerization of butylanoacrylate. The particles are coated with polysorbate, a penetration enhancer. Analgesic activity is obtained after intravenous administration. Unlike the present invention, however, the Schroder peptide must be chemically-bound to the polymeric material or to the polysorbate. The formulation is therefore a physical mixture of active drug and polymeric material.
Tsuzuki et al., Biochem. Pharm. Vol. 41, p. R5 (1991) teaches that analogues of leuenkephalin can be derivatized with adamantane moiety to obtain lipophilic enkephalin that shows an antinociceptive effect after subcutaneous administration. Modification at the N-terminus abolishes activity while the derivative at the C-terminus through ester bond retains activity. It is postulated that the activity is obtained after cleavage of the adamantane moiety. The derivative is therefore a prodrug, a concept not consistent with aspects of the present invention in which the-therapeutic conjugate retains the activity of the native peptide.
Prokai-Tatra, J. M. Chem, Vol. 39, p. 4777 (1996) teaches that a leucine-enkephalin analogue can be modified with chemical delivery system which is based on a retrometabolic drug design. The enkephaklin analogue is derivatized with a dihydropyridine moiety at the N-terminus and a lipophilic moiety at the C-terminus. After intravenous administration of the conjugate, analgesic response is observed. It is postulated that the lipophilic modification at the C-terminus enables penetration into the CNS, while the dihydropyridine moiety undergoes oxidative transformation to generate a charged moiety which restricts the peptides from effluxing into the circulatory system. Cleavage of the peptide from this moiety restores the observed analgesic activity. The derivatized peptide is inactive and regains activity only after metabolic transformation. The product is therefore a pure prodrug, requiring metabolic transformation to transform it into an active form.
U.S. Patent 4,933,324 to Shashoua teaches that certain natural fatty acids can be conjugated to neuroactive drugs. A highly unsaturated fatty acid of twenty-two (22) carbon chain length is particularly preferred. Administration of the conjugate shows absorption into the brain. As is the case with adamantane conjugation, this approach requires metabolic transformation of the prodrug conjugate of enkephalin to restore the activity of the enkephalin peptide.
There is therefore a compelling need in the art for pharmaceutically acceptable and effective therapeutic/diagnostic compositions capable of traversing the BBB without substantial loss or diminution of their therapeutic or diagnostic character.
The present invention broadly relates to therapeutic and/or diagnostic drug oligomer conjugates wherein a drug molecule is covalently bonded to an oligomer to form an amphiphilic conjugate. In one aspect, the oligomer comprises at least one lipophilic moiety and at least one hydrophilic moiety, and the size and nature of the amphiphilic and lipophilic moieties is so selected as to impart an amphiphilic nature to the resulting conjugate.
The present invention relates generally to amphiphilic drug-oligomer conjugates capable of traversing the BBB and to methods of making and using such conjugates.
In one aspect, the therapeutics are neuroactive drugs, proteins, peptides, and especially enkephalin analogues. The conjugates are stable in the environment of the bloodstream and resist degradation by the enzymes of the BBB and in the CNS. Furthermore, the conjugates readily traverse the BBB.
In one aspect, the drug oligomer conjugates produce their intended pharmacological effect without undergoing metabolic cleavage of the oligomer.
In another aspect the lipophile and hydrophile are connected by a labile, hydrolyzable bond. When the bond is hydrolyzed in the CNS, the hydrophile remains attached to the drug.
The amphiphilic oligomers are composed of lipophilic and hydrophilic moieties. The lipophilic moieties are preferably natural fatty aids or alkyl chains. Preferably, the fatty-acid moiety is a straight chain molecule having (saturated or unsaturated) carbon atoms and suitably ranges from four (4) to twenty six (26) carbon atoms. Most preferably, the fatty acid has from fourteen (14) to twenty two (22) carbon atoms.
The hydrophilic moieties are preferably small segment of polyethylene glycol (PEG), preferably having 1-7 PEG units, and more preferably 1-5 PEG units. The length and composition of the lipophilic moieties and the hydrophilic moieties may be adjusted to obtain desired amphiphilicity.
In another aspect, a cholesterol or adamantane moiety is substituted for straight chain fatty acid portion of the oligomers.
Examples of preferred oligomers are as follows:
CH3(CH2)n(OC2H4)mOHxe2x80x83xe2x80x83(Formula 1);
wherein n=3 to 25and m =1to 7;
CH3(CH2)n(OC2H4)mOCH2CO2Hxe2x80x83xe2x80x83(Formula 2);
wherein n=3 to 25 and m=1 to 16;
CH3(CH2)nCX(OC2H4)mOHxe2x80x83xe2x80x83(Formula 3);
wherein n=3 to 25, m=1 to 7 and X=O;
Rxe2x80x94(OC2H4)mCH2CO2Hxe2x80x83xe2x80x83(Formula 4)
wherein m=0 to 5 and R=cholesterol or adamantane; or
Rxe2x80x94OCO(C2H4O)mCH2CO2Hxe2x80x83xe2x80x83(Formula 5);
wherein m=0to 5;
CH3(CH2xe2x80x94CHxe2x95x90CH)6(CH2)2CH2(OC2H4)mOHxe2x80x83xe2x80x83(Formula 6);
wherein m=0 to 7;
CH3(CH2xe2x80x94CHxe2x95x90CH)6(CH2)2CX(OC2H4)mOHxe2x80x83xe2x80x83(Formula 7);
wherein m=1 to 7 and X=N or O.
Other unsaturated fatty acid moieties which can be used according to the present invention include oleic, linoleic, and linolenic.
For example, in one aspect, the lipophile and hydrophile are connected by hydrolyzable bonds. It is preferred to provide hydrolyzable bonds between the fatty acid and the hydrophilic moieties. This permits hydrolysis to occur after penetration into the CNS, thus releasing the active peptides with the hydrophilic group still attached to the peptide. As a result, the peptide acquires a more hydrophilic character and efflux to the circulatory system is thereby hindered.
Exemplary conjugates having non-hydrolyzable bonds are as follows: 
In another aspect, the lipophile and hydrophile are connected by hydrolizable bonds.
For example: 
In one aspect, the covalent bond between the oligomer and the drug is preferably amide (a carboxy group of the oligomer is linked to an amine group of the peptide drug), or carbamate an chloroformate group of the oligomer is linked to an amine group of the peptide drug). In general, the derivitizable amine group of the peptide is the amine of the N-terminus or a nucleophilic amino residue, usually found on the epsilon amino residue of a lysine residue.
In another aspect, an ester (a carboxy group of the peptide is covalently coupled to a hydroxyl group of the oligomer or a carboxy group of the oligomer is covalently coupled to a hydroxyl group of the drug), amide (a carboxy group of the oligomer is linked to an amine group of the drug) or carbamate (an chloroformate group of the oligomer is linked to an amine group of the drug) bond is provided for non peptide drugs.
For the enkephalin analogues, the preferred peptides are leu enkephalin lysine (SEQ ID NO:50) and met enkephalin lysine (SEQ ID NO:49). The amino acid side chain of the lysine is preferably utilized in bonding.
The amphiphilic drug-oligomer conjugate may comprise multiple oligomers of differing compositions.
In another aspect, the amphiphilic drug oligomer conjugates moieties are configured as follows:
Rxe2x80x94OCH2CH2OCH2CH2C(O)OCH2CH2CH2OCH2CH2CH2NH-Enkephalin (SEQ ID NO:1); or
Rxe2x80x94OCH2CH2OCH2C(O)OCH2CH2NH-Enkephalin (SEQ ID NO:1).
Wherein R=alkyl1-26, cholesterol or amantane.
In another aspect of the amphiphilic oligomer moieties are sugar moieties coupled to natural fatty acids and segments of polyethylene glycol. The PEG moiety serves to increase the amphiphilicity of the fatty sugar. Examples of arrangements including sugar moieties are provided in FIGS. 1A-1C.
In another aspect, PEG is used as a spacer group in the amphiphilic conjugate and the length and number of the PEG moieties can be varied to refine the amphiphilicity of the conjugate. Increasing the number of PEGs increases the hydrophilicity of the conjugate.
In another aspect of the invention, a proline or alanine is added to the N-terminus of the peptide. In a preferred aspect, a proline or alanine is added to the N-terminus of an enkephalin peptide and the oligomer moiety is coupled to the N-terminus of the proline or alanine residue.
After absorption into the central nervous system, the esters of the fatty sugar are hydrolysized leaving a hydrophilic moiety. Efflux is hindered and the brain aminopeptidases cleave the proline or alanine portion leaving the peptide to regain full activity.
The invention also provides a pharmaceutical composition comprising an amphiphilic drug oligomer conjugate and a pharmaceutically acceptable carrier.
In another aspect, a pharmaceutical composition is provided comprising (1) a mixture of an enkephalin conjugate according to the present invention wherein the enkephalin peptide has proline or alanine added to its N-terminus and an enkephalin conjugate according to the present invention which does not have a proline or alanine added to the N-terminus, and (2) a pharmaceutical carrier. This aspect provides a faster acting sustained dose of enkephalin.
The invention also provides methods of administering a conjugate of the invention.
The invention further provides assays, both in vitro and in vivo, for testing the efficacy of the conjugates of the invention.
Other objects and further scope of applicability of the present invention will become apparent from the detailed description given hereafter. It should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given byway of illustration only, since various changes and modifications will become apparent to those skilled in the art from the detailed description.
2.1 DEFINITIONS
As used herein, the term xe2x80x9clipophilicxe2x80x9d means the ability to dissolve in lipids and/or the ability to penetrate, interact with and/or traverse biological membranes.
As used herein, the term, xe2x80x9clipophilic moietyxe2x80x9d or xe2x80x9clipophilexe2x80x9d means a moiety which is lipophilic and/or which, when attached to another chemical entity, increases the lipophilicity of such chemical entity, e.g., fatty acid, cholesterol.
As used herein, the term xe2x80x9chydrophilicxe2x80x9d means the ability to dissolve in water.
As used herein, the term xe2x80x9chydrophilic moietyxe2x80x9d or xe2x80x9chydrophilexe2x80x9d refers to a moiety which is hydrophilic and/or which when attached to another chemical entity, increases the hydrophilicity of such chemical entity, e.g., sugars, PEG.
As used herein, the term xe2x80x9camphiphilicxe2x80x9d, means the ability to dissolve in both water and lipids.
As used herein, the term xe2x80x9camphiphilic moietyxe2x80x9d means a moiety which is amphiphilic and/or which when attached to a peptide or non peptide drug increases the amphiphilicity of the resulting conjugate, e.g., PEG-fatty acid oligomer, sugar fatty acid oligomer.
As used herewith, the term xe2x80x9cneuroactive drugxe2x80x9d is used broadly to encompass any peptide or other drug having an activity within the CNS, e.g., enkephalin, enkephalin analogues.
As used herein the term xe2x80x9cpeptidexe2x80x9d is intended to be broadly construed as inclusive of polypeptides per se having molecular weights of up to about 10,000, as well as proteins having molecular weights of greater than about 10,000.
As used herein, the term xe2x80x9ccovalently coupledxe2x80x9d means that the specified moieties are either directly covalently bonded to one another, or else are indirectly covalently joined to one another through an intervening moiety or moieties, such as a bridge, spacer, or linkage moiety or moieties.
As used herein, the term xe2x80x9cdrugxe2x80x9d means a substance intended for use in the diagnosis, characterization, cure, mitigation, treatment, prevention or allaying the onset of a disease, disease state, or other physiological condition or to enhance normal physiological functioning in humans and/or in non human animals.
For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the subsections which follow.
The present invention relates generally to amphiphilic drug oligomer conjugates capable of traversing the BBB and to methods of making and using such conjugates.
The drugs are preferably neuro-active drugs, proteins peptides and especially enkephalin analogues. The conjugates are stable in the environment of the bloodstream and resist degradation by the BBB. The conjugates readily traverse the BBB.
In one aspect, the conjugates produce their intended pharmacological effect without requiring metabolic cleavage of the oligomer. When cleavage of the oligomer occurs, the drug retains activity.
The amphiphilic oligomers are composed of lipophilic and hydrophilic moieties. The lipophilic moieties are preferably natural fatty acids or alkyl chains. The hydrophilic moieties are preferably small segments of PEG, having 1 to 7 PEG moieties, and preferably having 1 to 5 PEG moieties. The length and composition of the lipophilic moieties and the hydrophilic moieties may be adjusted to obtain desired amphiphilicity. For example, the carbon chains of the fatty acid or alkyl moieties may be lengthened to increase lipophilicity, while PEG moieties may be lengthened to increase hydrophilicity.
Preferably, the fatty acid moiety is a straight chain molecule having saturated and unsaturated carbons and ranges from four (4) to twenty-six (26) carbon atoms. Most preferably the fatty acid has from fourteen (14) to twenty two (22) carbon atoms.
A cholesterol or adamantane moiety can be substituted for straight chain fatty acid as the lipophilic portion of the oligomers.
Examples of preferred oligomers arenas follows:
CH3(CH2)n(OC2H4)mOHxe2x80x83xe2x80x83(Formula 1);
wherein n=3 to 25and m=1 to 7;
xe2x80x83CH3(CH2)n(OC2H4)mOCH2CO2Hxe2x80x83xe2x80x83(Formula 2);
wherein n=3 to 25 and m=1 to 6;
CH3(CH2)nCO(OC2H4)mOHxe2x80x83xe2x80x83(Formula 3);
wherein n=3 to 25, and m=1 to 7;
Rxe2x80x94(OC2H4)mCH2CO2Hxe2x80x83xe2x80x83(Formula 4)
wherein m=0 to 5and R=cholesterol or adamantane; or
Rxe2x80x94OCO(C2H40)mCH2CO2Hxe2x80x83xe2x80x83(Formula 5);
wherein m=0 to 5,
CH3(CH2Hxe2x80x94CHxe2x95x90CH)6(CH2)2CH2(OC2H4)mOHxe2x80x83xe2x80x83(Formula 6);
wherein m=1 to 7;
CH3(CH2xe2x80x94CHxe2x95x90CH)6(CH2)2CO(OC2H)mOHxe2x80x83xe2x80x83(Formula 7);
wherein m=1 to 7.
Other unsaturated fatty acid moieties which can be used according to the present invention include oleic, linoleic and linolenic.
In certain instances, it is preferred to provide hydrolyzable bonds between the polyethylene glycol and the fatty acid moieties. This permits hydrolysis to occur after penetration into the central nervous system thus releasing the active peptides with the polyethylene glycol group still attached to the peptide. The peptides acquire a more hydrophilic character and efflux to circulatory system is thereby hindered.